1. Field of the Invention
The present invention relates to the field of diagnostic tests for determining if a human male is infertile. More specifically, the present invention provides a method for identifying infertile males by assessing the ability of a particular human sperm sample to fertilize a human egg. The invention also relates to the field of screening protocols, as a method for screening human sperm samples for use in human fertilization is also provided. The invention also relates to the field of diagnostic kits, as a kit for detecting male infertility is also disclosed.
2. Description of the Related Art
Approximately one in six couples find themselves involuntarily infertile. This translates to between two and four million couples in the United States alone. Although numerous tests are available for diagnosing infertility problems, 16% of all couples that seek medical treatment are diagnosed with what is described clinically as "unexplained infertility". (J. A. Collins, P. J. Crosignani, Int. J. of Gyn. and Obstet., Vol. 39, p. 267-275 (1992)).
The term "unexplained infertility" is applied to virtually any clinically inexplicable failure of a male and female couple to conceive after extensive fertility testing of both partners reveals no identifiable cause for the couples infertility. After exhausting all available infertility tests with both male and female samples and other recognized infertility evaluations (i.e., female post-coital tests, timed endometrial biopsy, hysterosalpingogram, laparoscopy, male, "normal" sperm analysis, with sperm counts greater than 20 million/ml on at least two occasions, total sperm numbers of 40 million or more, sperm motility greater than 60%, and normal morphology in more than 60% of the sperm.sup.3, and the couple has had a history of involuntary infertility for at least 2 years, a human couple is diagnosed simply as "unexplainably infertile." Such couples historically undergo numerous invasive, protracted and expensive assisted reproductive technology attempts in their pursuit of pregnancy.
Tests of human sperm function have been developed in recognition that standard semen analysis provides a limited evaluation of the male's fertility potential. In fact, males having unexplained infertility are found to be normal in standard semen analysis. One such fertility test for human males is the sperm penetration assay (SPA) ..sup.38-40,70,71,82 The SPA tests the activation of a human sperm nucleus following entry into a fertilized egg by a determination of the percentage of eggs that are penetrated by the sperm (as determined by counting the eggs that contain decondensed or activated sperm nuclei). In general, this assay is designed to test the sperm's ability to get its nucleus into the egg. However, this protocol is not used nor may it be used to indicate the efficiency of the sperm decondensation process. During the SPA, the egg (for example, the commonly used hamster egg) routinely becomes bound with a large number of sperm that do not enter the egg and thus do not decondense. Thus, using this assay, one cannot tell a non-decondensed sperm (because it has become bound to the egg) apart from a sperm that has entered the egg and not decondensed as a result of not responding to the egg "activation" signals.
In some cases, the present inventor have observed a zero SPA score may not be reflecting the penetration capabilities of a particular sperm, but instead may reflect the sperm's inability to decondense.
Various techniques have been devised by researchers for using permeabilized sperm incubated in frog egg extracts to study sperm activation events. For example, using Xenopus laevis frog sperm incubated in Rana pipiens frog egg extract.sup.1 and using human sperm incubated in Xenopus laevis frog egg extract..sup.14-18 It has been observed that a normal (i.e., of proven human egg fertilizing capacity) human sperm nucleus becomes "activated" upon entry into a frog egg cytoplasm experimentally..sup.14-18
The "activation" of a normal human sperm has been reported in preparations of Xenopus laevis frog egg extract, which is reportedly attributable to the presence of "factors" in the frog egg extract itself..sup.14-18 These activation "factors" in Xenopus laevis frog egg extract have been the focus of much investigation by the present inventor and others, and have facilitated the general characterization of "activation" events of normal human sperm (such as the events of the sperm nuclear chromatin decondensation-recondensation cycle and DNA synthesis) ,.sup.14-18 as well as a "molecular" understanding of the "activation" events..sup.45-49,51-61,64
While the experimental model of Xenopus Laevis egg extract has been described which relates in general to the events of "normal" human sperm chromatin decondensation after activation, .sup.14-18 the efficiency of the decondensation process has not been described as pertinent to the sperm's fertilizing capacity. Nor is a diagnostic tool for determining or elucidating a particular cause of "unexplained" human male infertility using relative sperm "activation" events been characterized.
Additionally, analysis of the particular onset or duration of, for example, sperm decondensation or sperm recondensation, or the amount of, for example, DNA synthesis, indicators of human male infertility or human sperm capacity to fertilize a human egg, has not been described or suggested in any of the literature. The use of mammalian eggs as a means to obtain extract to follow the sperm activation events has not been possible because it is difficult, if not impossible, to obtain enough extract to examine mammalian fertilization..sup.43,44
Biological models for assessing human male fertility have not been established which employ sperm decondensation, recondensation and DNA synthesis events as a diagnostic tool. Moreover, the failure of currently available experimental and clinically accepted tests to examine the relative rate and degree of activity during sperm "activation events", as between "normal" human sperm and sperm with unknown fertilizing capacity, prevents the discovery of the differences which the present inventor have found definitive of the cause of some couples inability to successfully conceive.
An alternative method for diagnosing some cases of "unexplained infertility" through a system which was relatively non-invasive, and which provided for a rapid and accurate prognostic indicator of a human sperm samples' fertilizing capacity for a human egg, would save many currently "unexplained infertile" couples continued expense and invasive, and sometimes uncomfortable, attempts at pregnancy using assisted reproductive technologies.
Varicocele has been recognized as one of the leading causes of male infertility, and is frequently associated with abnormal sperm characteristics that include low values of sperm density, motility, and/or abnormal morphology, some also having abnormal SPA results. Steeno et al. recognizes three degrees of varicocele (grades I-III) and an additional group of clinically suspect cases.sup.50 Here, tortuous dilations of the testicular veins result in stagnation of venous blood in the pampiniform plexus. Several causes are suggested to explain the negative effects of varicocele on spermatogenesis; these include: (a) hyperthermia; (b) reflux of renal and adrenal venous blood into the testes; (c) altered testicular steroidogenesis; (d) hypoxia; and (e) mechanical compression..sup.62 However, none of these hypotheses are accepted unequivocally, and the mechanism of the pathophysiology associated with varicocele is still not well understood. A noninvasive technique for screening varicocele patients for the cause of their infertility would also provide a technique for screening potential candidates for varicocelectomy as a corrective measure for enhancing the fertility of the patient, as well as a technique for measuring the success of such surgeries in patients having undergone an elected varicocelectomy.
Methods for evaluating the potential antifertility effects of toxic agents to which human males are routinely exposed are also needed in the overall management.of fertility problems associated with human sperm malfunction. Such methods have not as yet been proposed in the medical arts.